Pathways that include cascades of serine proteases are important in the responses of vertebrate animals to bleeding and infection. Similar pathways have been identified in a few invertebrate animals. Experiments in this proposal are designed to test the hypothesis that activation of protease zymogens present in hemolymph is a key factor in stimulating several different types of insect responses to wounding and infection. Goals of the research are to identify and characterize proteases in hemolymph of Manduca sexta that participate in responses to wounding and infection, to identify their target substrates, and to gain an understanding of how such protease are activated and regulated. Responses to parasites that are mediated by hemolymph proteases may be important in determining the capacity of blood-sucking insects to act as vectors of human diseases. A long term objective of this research is to use the knowledge gained from these studies to investigate homologous molecules in vector insect species. The specific aims of the project are: 1. Identify and characterize serine proteases involved in activation of pro-phenol oxidase. Proteases linked to activation of pro-phenol oxidase will be purified and characterized. Amino acid sequence information and antibodies produced to these proteins will be used to screen cDNA libraries. Recombinant enzymes will be produced using a baculovirus expression system. The set of reagents produced (clones, antibodies, recombinant proteins, antisense oligonucleotides) will be used in experiments to work out the pathway that leads to activation of pro-phenol oxidase. 2. Identify protein substrates in hemolymph, which when cleaved, produce signals that activate expression of anti-microbial protein genes in fat body. This aim tests the hypothesis that recognition of bacteria or fungi in the hemocoel triggers activation of serine proteases, perhaps in a cascade, that result in specific cleavage of a plasma protein to produce a fragment that stimulates fat body to synthesize and secrete anti- bacterial and anti-fungal proteins. 3. Investigate the proteolytic activation of the precursor of Manduca paralytic peptide. A peptide from Manduca hemolymph induces rapid paralysis and stimulates plasmatocyte spreading and aggregation. This 23- residue peptide is apparently present in plasma as larger inactive precursor, which is rapidly converted by a specific proteolysis an active molecule upon bleeding. A cDNA for the precursor of one of this peptide will be cloned, the precursor will be produced as a recombinant protein, its activation by proteases from hemolymph will be investigated.